The mechanisms by which cell surface binding of insulin is regulated will be investigated by the studies proposed in this application. The role insulin-dependent receptor subunit dissociation plays in short-term modulation (negative cooperativity) will be evaluated. Investigation of the mechanism of long-term modulation (up or down regulation) will be directed at identifying and quantitating intracellular insulin receptor pools in an attempt to identify the major control point within the cell. Receptor subunit dissociation will be evaluated by quantitating the amount of insulin bound to each size binding species using both reversibly bound radioiodinated insulin and covalently bound radioiodinated insulin as markers. Sizing of the subunits will be done by gel filtration columns, sucrose gradient centrifugation, and polyacrylamide gel electrophoresis. Liver from normal mice with experimentally manipulated insulin levels, sulfonylurea or biguanide drug treatment, or phosphodiesterase inhibition by aminophylline will be fractionated. The fractionation scheme involves two differential sedimentation steps and final fractionation by a linear sucrose gradient. This technique allows demonstration of the relationship of various subcellular organelles to one another. Insulin receptor on the plasma membrane and within the cell will be quantitated using radioiodinated insulin binding and by an anti-insulin receptor immunoassay. It is anticipated that long-term down regulation of insulin receptors in this experimental model will be applicable to the regulation of the insulin receptor in human obesity and maturity onset diabetes mellitus. Understanding the mechanisms involved in modulation of insulin receptors may enable specific therapeutic maneuvers directed not at increasing insulin concentration at the cell but at increasing cell surface insulin receptor levels.